Neurotherapeutic treatment for sexual dysfunction

ABSTRACT

A method for improving sexual function is described. A mammal suffering from sexual dysfunction or otherwise in need of enhanced sexual function is administered a compound selected from those that are capable of inhibiting the activity of β-lactams, penicillin-binding protein, carboxypeptidase. Such compounds, including particularly β-lactam ring-containing compounds, can be used to formulate pharmaceutical formulations useful for improving sexual function.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C §119(e) to U.S.Provisional Application Ser. No. 60/299,060, filed on Jun. 18, 2001.

FIELD OF THE INVENTION

This invention relates to a method for improving sexual function. Moreparticularly, this invention is directed to the use of compounds capableof exhibiting specific binding affinity to and inhibiting the activityof certain bacterial enzymes and structurally related mammalian enzymesfor improving sexual function and for reducing or eliminating theindicia of sexual dysfunction in a mammal either suffering from suchdisability or exposed to conditions tending to engender such disability.

BACKGROUND OF THE INVENTION

Sexual dysfunction is characterized by a disturbance in the processesthat are involved in the sexual response cycle or by pain associatedwith sexual intercourse. The sexual response cycle comprises the fourphases of desire, excitement, orgasm and resolution. Disorders of sexualresponse may occur at one or more of these phases. The sexualdysfunctions include sexual desire disorders, sexual arousal disorders,orgasmic disorders and sexual pain disorders. Sexual dysfunctions causemarked distress and interpersonal difficulty. While progress has beenmade in the treatment of such disorders, there remains significant needfor alternative therapeutic approaches.

A normal erection occurs as a result of a coordinated vascular event inthe penis. This is usually triggered neurally and consists ofvasodilation and smooth muscle relaxation in the penis and its supplyingarterial vessels. Arterial inflow causes enlargement of the substance ofthe corpora cavernosa. Venous outflow is trapped by this enlargement,permitting sustained high blood pressures in the penis sufficient tocause rigidity. Muscles in the perineum also assist in creating andmaintaining penile rigidity. Erection may be induced centrally in thenervous system by sexual thoughts or fantasy, and is usually reinforcedlocally by reflex mechanisms. Erectile mechanics are substantiallysimilar in the female for the clitoris. Impotence or male erectiledysfunction is defined as the inability to achieve and sustain anerection sufficient for intercourse. Impotence in any given case canresult from psychological disturbances (psychogenic), from physiologicalabnormalities in general (organic), from neurological disturbances(neurogenic), hormonal deficiencies (endocrine) or from a combination ofthe foregoing. These descriptions are not exact, however. There iscurrently no standardized method of diagnosis or treatment. As usedherein, psychogenic impotence is defined as functional impotence with noapparent overwhelming organic basis. It may be characterized by anability to have an erection in response to some stimuli (e.g.,masturbation, spontaneous nocturnal, spontaneous early morning, videoerotica, etc.) but not others (e.g., partner or spousal attention).Various methods for the treatment of impotence have been suggested,including external devices, for example, tourniquets (see U.S. Pat. No.2,818,855). In addition, penile implants, such as hinged or solid rodsand inflatable, spring driven or hydraulic models, have been used forsome time. The administration of erection effecting and enhancing drugsis taught in U.S. Pat. No. 4,127,118 to LaTorre. That patent teaches amethod of treating male impotence by injecting into the penis anappropriate vasodilator, in particular, an adrenergic blocking agent ora smooth muscle relaxant to effect and enhance an erection. Morerecently, U.S. Pat. No. 4,801,587 to Voss et al. teaches the applicationof an ointment to relieve impotence. The ointment consists of thevasodilators papaverine, hydralazine, sodium nitroprusside,phenoxybenzamine, or phentolamine and a carrier to assist absorption ofthe primary agent through the skin. U.S. Pat. No. 5,256,652 toEl-Rashidy teaches the use of an aqueous topical composition of avasodilator such as papaverine together withhydroxypropyl-.beta.-cyclodextrin. Sexual functions in females can bedivided into several broad areas: desire, arousal, and orgasm. Studieshave indicated that up to 63% of women exhibit dysfunctions in eitherarousal or orgasmic stages of sexual activities (Frank E, et al., 1978.N Engl J Med 299: 111). Sexual disorders such as dyspareunia andvaginismus, reduce the arousal phase of female sexual functioning.Impaired clitoral responsiveness can lead to orgasmic disorders. Theprevalence of female sexual dysfunction increases with age (Goldstein Mand Teng N. 1991, Clin Geriatr Med 7:41; Thirlaway K et al., 1996.Quality of Life Res 5:81; Slob A et al., 1990, J Sex Martial Ther16:59). Vascular risk factors of coronary diseases also increase theprobability of sexual dysfunction in postmenopausal females(Sadeghi-Nejad H et al., 1996, J Urol 155:677A). Female sexualdysfunction can be due to an impairment in endothelium dependentvasodilation and smooth muscle relaxation which in turn could lead toimpairment of vascular dependent events associated with sexualfunctioning. During sexual arousal, an increase in vaginal blood flowoccurs which in turn results in vaginal lengthening and enhancedproduction of vaginal fluid. Enhanced clitoral blood flow occurs duringarousal leading to clitoral engorgement and erection. Impairment ofthese vascular dependent events can lead to impairment in vaginallubrication and/or a diminution in vaginal enlargement during thearousal stage of female sexual function. An abnormality in thesevascular dependent events could impair the arousal and/or orgasmicphases of sexual functioning.

SUMMARY OF THE INVENTION

The present invention provides a unique therapeutic approach toimproving sexual function or the treatment of sexual dysfunction by whatis presently believed to be a mechanism comprising inhibition of one ormore neurogenic peptidases and a consequent therapeutically beneficialchange in the concentrations of multiple neurologically significantneurotransmitters in the brain.

The method comprises the step of administering to a mammal sufferingfrom or disposed to develop sexual dysfunction, or otherwise in need ofenhanced sexual function, a compound which exhibits specific bindingaffinity to, and which inhibits function, of an enzyme selected from agroup consisting of β-lactamase, penicillin-binding protein, andcarboxy-peptidase in an amount effective to promote normal or enhancedsexual function of said mammal. The compound should possess sufficientblood-brain barrier transport properties so that blood levels of saidcompound achieved by any one of a wide variety of routes ofadministration, can provide a concentration of said compound in thecentral nervous system of the mammal undergoing treatment effective toimprove sexual function, either by inhibiting the activity of one ormore neurogenic enzymes, for example, carboxy peptidases and/or byanother yet undefined mechanism of action. Treatment is effective inmales, evidenced by enhanced erectile function, and in femalesevidenced, for example, by enhanced solicitation behavior.

In one embodiment of the invention the compound is a β-lactamring-containing compound which is capable of inhibiting the biologicalactivity of a β-lactamase, a penicillin-binding protein, or a carboxypeptidase such as carboxy peptidase E. In one embodiment the compound isclavulanic acid or a pharmaceutically acceptable salt or ester thereof.Other compounds recognized for their β-lactamase activity, theirantibiotic activity, and/or their ability to inhibit carboxypeptidase Eor other neurologically significant carboxy peptidases and having therequisite threshold blood-brain barrier transport property can beemployed beneficially in accordance with the present method. Suchcompounds can be administered by any one of a wide variety ofart-recognized routes of administration, including but not limited tooral ingestion, or parenteral, transdermal, inhalation, sublingual orbuccal administration. Dosage ranges depend on the patientcondition/circumstances and the pharmacologically significant propertiesof the therapeutic compound including, for example, minimum inhibitoryconcentration, absorption, protein binding, blood-brain barriertransport and the like, and dosage levels can be determined byextrapolation of effective concentrations in test animals. Typically thecompounds are administered in accordance with this invention one to fourtimes a day at a dose of about 0.01 to about 10 mg/kg, about 0.1 toabout 10 mg/kg, or up to about 0.2 mg/kg to about 10 mg/kg. Unit dosageforms can be prepared to contain about 1 mg to about 500 mgs of theβ-lactam compound in combination with a pharmaceutically acceptablecarrier determined typically by the targeted route of administration.Optionally the β-lactam compound can be formulated in a prolongedrelease dosage form from which effective amounts of the compound arereleased over a period of about three hours to about one week or more.Methods for preparing such controlled release dosage forms are wellknown in the art and can be applied to formulate the compounds for usein accordance with this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing anxiety scores for clavulanic acidadministered to cotton-top tamarins.

FIG. 2 is a bar graph showing the effect of clavulanic acid on sexualarousal in cotton-top tamarins.

FIG. 3 illustrates several chromatograms from HPLC electrochemicaldetection of dialysate samples collected in the nucleus accumbens in atest animal following intraperitoneal administration of 10 μg/kg ofclavulanic acid. Retention times for norepinephrine (NE), epinephrine(Epi), dopamine metabolite DOPAC, dopamine, serotonin metabolite HIAA,serotonin (5-HT), and homovanillic acid (HVA) are indicated.

FIGS. 4A and 4B are graphic representations of the concentrations ofneurotransmitters in the nucleus accumbens of a test animal (AC-1) as afunction of time from administration of clavulanic acid.

FIG. 4B is similar to FIG. 4A except that it graphical depicts therelative concentration of serotonin and serotonin metabolites in thenucleus accumbens as a function of time post administration ofclavulanic acid.

FIGS. 5A and 5B are similar to FIGS. 4A and 4B but the data is that froma second test animal (AC-2)

FIGS. 6A and 6B are similar to FIGS. 4A and 4B except they depict testdata from a third test animal (AC-3)

FIGS. 7A and 7B are similar to FIGS. 4A and 4B except that they depicttest data from a fourth test animal (AC-4)

FIGS. 8A and 8B are also similar to FIGS. 4A and 4B except that theydepict test results from a fifth test animal (AC-5).

DETAILED DESCRIPTION OF THE INVENTION

There is provided in accordance with this invention a method forimproving sexual function in a mammal suffering from or disposed todevelop sexual dysfunction or otherwise in need of enhanced sexualfunction to promote normal or enhanced sexual function. Sexualdysfunction can be the result of any one or a combination of a widevariety of psychological or physiological patient conditions.Alternatively, sexual dysfunction can be a consequence of thetemporal/local environment and physiological and psychological stressimposed by same. It is a condition that affects not only humans but alsonon-human mammals such as farm animals or zoo animals maintained incaptivity.

As used herein, the term “sexual dysfunctions” includes sexual desiredisorders, sexual arousal disorders, orgasmic disorders, sexual paindisorders, sexual dysfunction due to a general medical condition,substance-induced sexual dysfunction and sexual dysfunction nototherwise specified. These sexual dysfunctions may be further defined bythe nature of the onset of the disorder: either lifelong type oracquired type; by the context in which the disorder occurs: eithergeneralized type or situational type; and by the etiological factorsassociated with the disorder: either due to psychological factors or dueto combined factors. Specifically, sexual desire disorders includehypoactive sexual desire disorder and sexual aversion disorder. Sexualarousal disorders include female sexual arousal disorder and maleerectile disorder. Orgasmic disorders include female orgasmic disorder,male orgasmic disorder and premature ejaculation. Sexual pain disordersinclude dyspareunia and vaginismus. Sexual dysfunctions due to a generalmedical condition may result from neurological conditions (e.g. multiplesclerosis, spinal cord lesions, neuropathy and temporal lobe lesions),endocrine conditions (e.g. diabetes melitus, hypothyroidism, hypogonadalstates and pituitary dysfunction), and vascular conditions andgenitourinary conditions (e.g. testicular disease, Peyronie's disease,urethral infections, postprostatectomy complications, genital injury orinfection, atrophic vaginitis, infections of the vagina and externalgenitalia, postsurgical complications such as episiotomy scars,shortened vagina, cystitis, endometriosis, uterine prolapse, pelvicinfections and neoplasms). Substance-induced sexual dysfunction canoccur in association with intoxication with the following classes ofsubstance: alcohol; amphetamine (and amphetamine-like substances);cocaine; opioids; sedatives, hypnotic and anxiolytics; and other unknownsubstances. A decrease in sexual interest and orgasmic disorders mayalso be caused by prescribed medication including antihypertensives,histamine H₂-receptor antagonists, antidepressants, neuroleptics,anxiolytics, anabolic steroids, and antiepileptics. Painful orgasm hasbeen reported with fluphenazine, thioridazine and amoxapine. Priapismhas been reported with the use of chlorpromazine, trazodone andclozapine, and following penile injections of papaverine orprostaglandin Selective serotonin reuptake inhibitors may causedecreased sexual desire or arousal disorders.

Also, as used herein, the term “sexual dysfunctions” includes any of theaforementioned sexual dysfunctions, including loss of libido, resultingfrom other medical conditions, most especially resulting from depressionand/or anxiety. As used herein, the term “treatment” refers both to thetreatment to promote normal or enhanced sexual function and to theprevention or prophylactic therapy of the aforementioned conditions.

In a related aspect of the invention it has been found thatadministration of β-lactamase inhibitors and other compounds capable ofinhibiting penicillin-binding protein and structurally related mammalianenzymes activate serotonin and/or dopamine neurotransmission in thebrain. Thus in accordance with another embodiment of the invention thereis provided a method of activating serotonin and/or dopamineneurotransmission in the brain of a mammal. The method comprises thestep of administering to said mammal a serotonin and/or dopamineneurotransmission enhancing amount of a compound selected from aβ-lactamase inhibitor, a penicillin sulfoxide, a penicillin sulfone, anda cephalosporin or a cephalosporin analog or derivative of the formula

wherein X is S, SO, SO₂′O, CR₂, R₃, wherein R₂ and R₃ are independentlyhydrogen or C₁-C₄ alkyl, R is hydrogen, a salt forming group or anactive ester fanning group; R¹ is hydrogen or C₁-C₄ alkoxy, T is C₁-C₄alkyl, halo (including chloro, fluoro, bromo and iodo), hydroxy,O(C₁-C₄) alkyl, or —CH₂B wherein B is the residue of a nucleophile B:H,and acyl is the residue of an organic acid Acyl OH.

In still another related aspect of the invention there is provided amethod for using such compounds for the preparation of pharmaceuticalformulations useful for activating serotonin and/or dopamineneurotransmission in the brain. Such formulations are expected to beeffective in the treatment of numerous disease states having aneurological dysfunction etiology. Such disease states include but arenot limited to addiction, obesity, and schizophrenia.

In still one other embodiment of the invention there is provided amethod for improving sexual function in a mammal suffering from ordisposed to develop sexual dysfunction or otherwise in need of enhancedsexual function. The method comprises the step of administering to saidmammal a compound selected from the group consisting of a β-lactamaseinhibitor, a penicillin, a penicillin sulfoxide, a penicillin sulfone,and a cephalosporin or a cephalosporin analog of the formula

wherein X is S, SO, SO₂′O, CR₂, R₃, wherein R₂ and R₃ are independentlyhydrogen or C₁-C₄ alkyl, R is hydrogen, a salt fanning group or anactive ester forming group; R¹ is hydrogen or C₁-C₄ alkoxy, T is C₁-C₄alkyl, halo (including chloro, fluoro, bromo and iodo), hydroxy,O(C₁-C₄) alkyl, or —CH₂B wherein B is the residue of a nucleophile B:H,and acyl is the residue of an organic acid Acyl OH.

The method can be utilized in both male and female patients and ispreferably carried out with compounds as specified that are withoutclinically significant antimicrobial activity, more preferably compoundsthat are substantially devoid of antimicrobial activity.

One group of compounds useful in accordance with the present inventionare β-lactamase inhibitors. There are many compounds that are reportedin the literature to exhibit the capacity to inhibit bacterialβ-lactamase activity. Such is typically measured by the compound'sability to inhibit the rate of hydrolysis of a penicillin orcephalosporin substrate by 50%, either with or without preincubation.Techniques for assessing or assaying β-lactamase inhibition andinhibition of other enzyme activity are well known in the art.

Most known β-lactamase inhibitors are compounds which themselvescomprise a β-lactamase ring structure. Both the patent and non-patentart are replete with reference to such compounds, their preparation, andtheir mechanism of action. Inhibition of bacterial β-lactamase can occureither by an irreversible mechanism or via a reversible mechanisminvolving a transient inhibited intermediate in which the β-lactamaseinhibitor binds to and thus blocks the active site on the β-lactamasemolecule. β-lactamases can be inhibited irreversibly by a β-lactamaseinhibitor which competitively or preferentially binds to the active siteon the β-lactamase molecule where it effectively acylates theβ-lactamase as a first step in deactivating the enzyme.

Exemplary of known β-lactamase inhibitors in commercial use areclavulanic acid, sulbactam, and tazobactam. Other known β-lactamaseinhibitors include derivatives or analogs of clavulanic acid includingdeoxyclavulanic acid, isoclavulanic acid, 9-deoxyclavulanic acid,9-amino deoxyclavulanic acid, and other clavulanic acid derivatives suchas those wherein the 9-hydroxy group has been chemically modified (e.g.as an acetate, n-methyl carbamate, methyl ether, benzyl ether, orthiomethyl ether). Sulbactam has been used to prepare prodrugs, forexample, sultamacillin which is absorbed from the gastrointestinal tractand then hydrolyzed into sulbactam and ampicillin. Other known β-lactamcontaining compounds known to possess β-lactamase inhibitor propertiesinclude olivanic acids and thienamycin of the carbapenem family of novelnaturally occurring β-lactam antibiotics and sultamicillin andaztreonam.

One preferred β-lactamase inhibitor for use in accordance with thepresent invention is clavulanic acid. It has only weak, though broadspectrum antibacterial activity, and it has a long record of safe use asa β-lactamase inhibitor in commercially available combinations withamoxycillin and ticarcillin. Moreover, it exhibits good oral adsorptionand transport across the blood-brain barrier into the cerebral spinalfluid. β-lactamase inhibitors can be administered in accordance withthis invention as their pharmaceutically acceptable salts or asbioactive esters which hydrolyze to provide therapeutic concentrationsof the β-lactamase inhibitor upon patient administration.

Effective dosages of the β-lactamase inhibitors when used in accordancewith the method of this invention depends on patient condition and themethod of administration. Animal tests indicate that clavulanic acid iseffective when administered intraperitoneally at a dose of about 1 μg/kgto about 50 μg/kg. Parenteral doses of β-lactamase inhibitors when usedin accordance with this invention range from about 0.02 to about 20mg/kg. Oral dosage levels are typically higher, ranging from about 0.05mg/kg to about 50 mg/kg. The dosage levels can be adjusted higher orlower by the attending physician depending on patient condition and theobserved clinical response to the initial dosage. Treatment inaccordance with this invention typically includes one to four dailydoses of β-lactamase inhibitor. Formulation of the inhibitor intocontrolled release dosage forms (either for parenteral or oral use)enables effective once or twice a day dosage protocols.

In addition to β-lactamase inhibitors, other β-lactam-containingcompounds, i.e., compounds having a β-lactam ring system, can be used inaccordance with this invention, generally to activate or enhanceserotonin and dopamine neurotransmission, and more specifically toimprove sexual function or to treat certain other disease statesresponsive to activation of serotonin and dopamine neurotransmission.

Examples of such compounds are β-lactam antibiotics, such as penicillinsand cephalosporins and derivatives, for example, their sulfoxides orsulfones, and analogs thereof, such as the art recognized1-oxa-1-dethiacephems and the 1-carba-1-dethiacephems. The prior art isreplete with reference to many of such compounds and their method ofsynthesis/preparation.

Penicillin sulfoxides or sulfones useful in this invention are of thegeneral formula

wherein X is SO or SO₂ and R and Acyl are as defined above.

The cephalosporin/cephalosporin analogs/derivatives useful in accordancewith this invention include both 2-cephem compounds of the formula

and 3-cephem compounds of the formula

wherein X is S, SO, SO₂′O, CR₂, R₃, wherein R₂ and R₃ are independentlyhydrogen or C₁-C₄ alkyl, R is hydrogen, a salt forming group or anactive ester forming group; R¹ is hydrogen or C₁-C₄ alkoxy, T is C₁-C₄alkyl, halo (including chloro, fluoro, bromo and iodo), hydroxy,O(C₁-C₄) alkyl, or —CH₂B wherein B is the residue of a nucleophile B:H,and Acyl is the residue of an organic acid Acyl OH.

Variations of substituents “Acyl,” “R¹” and “T” have been the subject ofyears of cephalosporin research which produced many commerciallysignificant cephalosporin antibiotics. While the nature of suchsubstituent can impact the biological activity of the respectivecompounds, the nature of such substituents is not the focus of thepresent invention.

One group of compounds for neurotherapeutic use herein are β-lactamantibiotics including penicillins, cephalosporins and monocyclic andbicyclic analogs or derivatives thereof. Commercially availableantibiotics for use in the present method and use include penams,cephems, 1-oxa-1-dethia cephems, clavams, clavems, azetidinones,carbapenams, carbapenems and carbacephems.

In one embodiment of this invention the β-lactam ring containingcompounds for us in this invention are without clinically significantantibiotic activity, and ideally, they are substantially devoid ofbiological activity. Such compounds include, for example, the 2-cephemcompounds generally and as well the 2- and 3-cephem sulfoxide (X═SO) andsulfone derivatives (S—SO₂) and penicillin sulfoxide and sulfonederivatives.

The β-lactam compounds for use in this invention having a carboxylatefunctional group can be administered as its pharmaceutically acceptablesalt or in the form of an in vivo hydrolysable (active) ester group.

Examples of suitable in vivo hydrolysable (active) ester groups include,for example, acyloxyalkyl groups such as acetoxymethyl,pivaloyloxymethyl, β-acetoxyethyl, β-pivaloyloxyethyl,1-(cyclohexylcarboonyloxy) prop-1-yl, and (1-aminoethyl)carbonyloxymethyl; alkoxycarbonyloxyalkyl groups, such asethoxycarbonyloxymethyl and alpha-ethoxycarbonyloxyethyl;dialkylaminoalkyl groups, such as ethoxycarbonyloxymethyl andβ-ethoxycarbonyloxyethyl; dialkylaminoalkyl especially di-loweralkylamino alkyl groups such as dimethylaminomethyl, dimethylaminoethyl,diethylaminomethyl or diethylaminoethyl:2-(alkoxycarbonyl)-2-alkenylgroups such as 2-(isobutoxycarbonyl) pent-2-enyl and2-(ethoxycarbonyl)but-2-enyl; lactone groups such as phthalidyl anddimethoxyphthalidyl; and esters linked to a second β-lactam compound.

Suitable pharmaceutically acceptable salts of β-lactam compounds used inthis invention include metal salts, e.g. aluminum, alkali metal saltssuch as sodium or potassium, alkaline earth metal salts such as calciumor magnesium, and ammonium or substituted ammonium salts, for examplethose with lower alkylamines such as triethylamine, hydroxy-loweralkylamines such as 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine ortris-(2-hydroxyethyl)amine, cycloalkylamines such as dicyclohexylamine,or with procaine, dibenzylamine, N,N-dibenzylethylenediamine,1-ephenamine, N-methylmorpholine, N-ethylpiperidine,N-benzyl-β-phenethylamine, dehydroabietylamine,N,N′-bisdehydro-abietylamine, ethylenediamine, or bases of the pyridinetype such as pyridine, collidine or quinoline, or other amines whichhave been used to form salts with known penicillins and cephalosporins.Other useful salts include the lithium salt and silver salt.

The amount of β-lactam compounds used to form the pharmaceuticalcomposition is that amount effective to provide upon delivery by theintended route of administration, an effective concentration of thecompound in neuronal tissue. Typically they are administered at a doseof about 0.01 mg/kg to about 10 mg/kg. Parenteral dosage forms typicallycan contain about 0.5 to about 50 mg/dose or 2- to 3-fold that amountwhen formulated in a controlled release parenteral dosage form, whileoral dosage fauns can typically contain about 1 to about 200 mg of theactive compound.

Compounds for therapy in accordance with this invention beformulated/combined with one or more pharmaceutically acceptablecarriers and may be administered, for example, orally in such forms astablets, capsules, caplets, dispersible powders, granules, lozenges,mucosal patches, sachets, and the like. In such formulations a theactive compound is combined with a pharmaceutically acceptable carrier,for example starch, lactose or trehalose, alone or in combination withone or more formulation excipients and pressed into tablets or lozengesor filled into capsules. Optionally, dosage forms intended for oralingestion administration such as tablets, caplets or capsules can beenterically coated to minimize hydrolysis/degradation in the stomach. Inanother embodiment the dosage form is formulated for oraladministration, and is formed as a prolonged release dosage form usingart-recognized formulation techniques for release the compound over apredetermined period of time.

Topical dosage forms, including transdermal patches, intranasal andsuppository dosage unit formulations containing the active compound andconventional non-toxic pharmaceutically acceptable carriers, adjuvantsand vehicles adapted for such routes of administration can also be used.

The pharmaceutical compositions suitable for injectable use inaccordance with this invention include sterile aqueous solutions ordispersions and sterile powders or lyopholysates for the extemporaneouspreparation of sterile injectable solutions or dispersions. The dosageforms must be sterile and it must be stable under the conditions ofmanufacture and storage. The carrier for injectable formulations istypically water but can also include ethanol, a polyol (for example,glycerol, propylene glycol and liquid polyethylene glycol), mixturesthereof, and vegetable oil.

Parenteral dosage forms useful in accordance with the present inventioncan also be formulated as injectable prolonged release formulations inwhich the active compound is combined with one or more natural orsynthetic biodegradable or biodispersible polymers such ascarbohydrates, including starches, gums and etherified or esterifiedcellulosic derivatives, polyethers, polyesters, polyvinyl alcohols,gelatins, or alginates. Such dosage formulations can be prepared forexample in the form of microsphere suspensions, gels, or shaped polymermatrix implants that are well-known in the art for their function as“depot-type” drug delivery systems that provide prolonged release of thebiologically active components. Such compositions can be prepared usingart-recognized formulation techniques and designed for any of a widevariety of drug release profiles.

Screening Clavulanic Acid (CLAV) for Anxiolytic Activity in Non-HumanPrimates

The cotton-top tamarin (Saguinus oedipus) is listed as an endangeredspecies with only 1-3 thousand remaining in the rain forests ofColombia. This monkey has a high stress temperament, making it difficultto breed and rear in captivity (Snowdon et al., 1985). Captive tamarinshave the highest prevalence of stress-induced colitis and colon cancerof any monkey studied (Clapp et al., 1988). The stress of captivitycontributes to the onset of inflammatory bowl disease since thiscondition is extremely rare in wild populations (Wood et al., 1998; Woodet al., 2000) and remission occurs when captive monkeys are returned tothe environmental conditions of the natural habitat (Wood et al., 1995).If orally administered CLAY could reduce anxiety and stress in thesenon-human primates there is a strong likelihood the drug would beeffective when tested in humans. Dr. Charles Snowdon, Department ofPsychology, University of Wisconsin at Madison was enlisted to design astudy using tamarins to test the anxiolytic activity of CLAY. Dr.Snowdon is a world expert in tamarin behavior with over twentypeer-reviewed publications in the field. Dr. Snowdon has studiedtamarins in the wild (Savage et al., 1997) and in the semi-naturalenvironment of his labs at Madison (Ginther et al., 2000).

Methods

Eight male/female pairs of tamarins with long standing pair bonds weretested following oral treatments of CLAV and vehicle (VEH) control.There were no offspring since males were vasectomized. Both members of apair received the same treatments at the same times. Each animal servedas its own control being tested both with CLAV or VEH one week apart.The treatment schedule was counter-balanced. CLAV was dissolved in waterand dispensed onto a small cookie in a volume of 100 μl. A single CLAVcookie was given to each member of a pair at an approximate dose of 1mg/kg body weight. Animals were given three CLAV cookies each day fortwo consecutive days. The 1^(st) cookie was given at 8:00 AM prior tothe morning feeding, the 2^(nd) at 11:00 AM prior to the noon feedingand the 3^(rd) at 2:00 PM prior to the late afternoon snack. All animalswere scored for anti-anxiety activity 60 min after the 3^(rd) treatmenton the second day.

This treatment regimen of three doses each day for two consecutive dayswas chosen to acclimate the animals to the treatment procedure (day 1)and to achieve steady-state blood concentration before testing (day 2).A pharmacokinetic assessment of CLAV was run by IVEDCO (Irving Tex.)which estimated an oral dose of 1 mg/kg every three hrs would producesteady-state levels yielding an average plasma concentration of 2.5μg/ml at the time of testing. Since the CSF/plasma ratio is 0.25 theestimated concentration in the brain would be around 0.3 μM. Given therate of clearance of CLAV, treatments from day 1 should not havecontributed to blood levels of drug on day 2.

Two observers blind to the treatment independently scored behavioralactivity. To elicit anxious/stressful behavior a novel object was placedinto the home cage. A different novel object was used for each testsession and the object presentation was counter-balanced. Following thepresentation of the novel object animals were scored for a duration of15 min for: 1) latency to approach and latency to touch the object, 2)time spent in contact or proximity with the object, 3) face and headmovement (frowns, head shakes and head cocks that reflect tension), 4)scratching (a validated measure of anxiety in macaques), 5) piloerection(autonomic stress response), and 6) visual scanning (nervous vigilance).On the first test session, it was noted that males treated with CLAVshowed a high incidence of penile erection, hence the number oferections; mounts and female solicitations were also scored.

Results

There was no significant difference between treatments in latency toapproach or latency to touch the novel object. Neither was there anydifference in time spent in proximity to the object. However, using thecombined measures of anxious/nervous behaviors (3, 4, 5 & 6) there weresignificantly fewer of these behaviors following treatment with CLAV(12.7+4.8) than with VEH (16.8=6.4) (Wilcoxon t=28, n=15 p<0.05) (FIG.1). There were no gender differences in drug response.

The unexpected result was the increase in erections by males. All eightmales showed at least one erection in 15 min when treated with CLAVwhile only three of these eight showed erections with VEH (FIG. 2). Themean was 2.25 erections in 15 min with CLAV and 0.37 with VEH.Cotton-top tamarins in captivity show an average base rate of 1.5erections per hour during their active diurnal period, a rate comparableto VEH treatment (Snowdon personal communication). Animals treated withCLAV show a rate of 9.0 erections per hour. These results weresignificant by a Wilcoxon test (t=0, n=7, p<0.02). Two of the eightfemales showed solicitation behaviors with CLAV treatment while nofemales showed solicitation with VEH. There was no significantdifference in mounting behavior between CLAV and VEH treatments.

Summary

Clavulanic acid given orally at a dose of 1 mg/kg reduced measures ofanxiety in male/female pairs of cotton-top tamarins. The time course ofaction was very rapid and appeared in less than two days of treatments.It is possible the effect could have been observed in 60 min after asingle dose of CLAV, as is the case in rodent studies. Studies arepresently underway to ascertain the minimal oral dose of CLAV and theshortest time course that significantly reduces anxiety in tamarins.Nonetheless, the time-course noted in these studies is far better thanthe selective serotonin reuptake inhibitors (SSRIs), e.g., fluoxetineand sertraline that are becoming more prevalent in the treatment ofanxiety disorders. SSRIs take several days to weeks before achievingclinical efficacy. In addition, the SSRIs suppress libido contributingto sexual dysfunction (Rothschild 2000). CLAV not only reduced anxietyin cotton-top tamarins, but it also increased sexual arousal asindicated by the increased rate of penile erections. The mechanism forthis biological effect is unknown. CLAV could have a direct psychogeniceffect on libido or the enhanced sexual arousal could be secondary to areduction in stress. The latter hypothesis is not unreasonable sincestress is one of the major factors contributing to sexual dysfunction(Smith 1988). The anxiety disorder, PTSD or post-traumatic stressdisorder has a particularly high incidence of sexual dysfunction (Kotleret al., 2000). Studies in rats show that long-term psychological stressimpairs sexual behavior, a result associated with a decrease inmonoamine activity in the brain (Sato et al., 1996). Enhancing monoamineactivity restores normal sexual behavior following chronic stress. Itwas determined to gather data to assess whether CLAV is increasingsexual arousal in tamarins by increasing monoamine activity in thebrain.

Using CLAV to reduce anxiety and enhance sexual arousal in thecotton-top tamarin, a species whose existence is jeopardized by its highstress temperament, is very significant. This drug may have a role inanimal husbandry to help in the breeding and rearing of endangeredspecies held in captivity. More importantly, the fact CLAV hasanxiolytic activity in the tamarin holds the promise the CLAY and otherβ-lactamase inhibitors and other β-lactam compounds may be an effectivetherapeutic for the treatment of anxiety disorders in humans and evenstress-related gastrointestinal disease.

While CLAY does not appear to bind to any of the well known signalingreceptors or transporters, is clearly altering brain chemistry in someway, either by neurogenic enzyme inhibition or by interaction with astill unrecognized receptor system, to achieve anxiolytic activity. Totest this hypothesis, extracellular neurotransmitter levels in the areaof the nucleus accumbens were assessed with microdialysis following CLAYtreatment. The accumbens is part of the limbic forebrain best know forits association with the pathophysiology of schizophrenia and drugaddiction but also thought to be involved in sensitization to early lifetrauma leading to the anxiety disorder PTSD or post traumatic stressdisorder (Charney and Bremner 1999).

Experimental Procedure

Ten male, Sprague Dawley rats were anesthetized with sodiumpentobarbital (50 mg/kg) and implanted with a unilateral microdialysisguide cannula aimed at the nucleus accumbens. Two days after recoveryfrom surgery a microdialysis probe (2 mm) was lowered to the area andconnected to an infusion pump through Tygon tubing. The dialysate wasartificial CSF at pH 7.4 delivered at a flow rate of 2*l/min. The first120 min of dialysate will be discarded. Thereafter, samples werecollected at 30 min intervals prior to and following IP CLAV treatment(10*g/kg). Samples were collected into microfuge tubes containing 5*l of0.16 N perchloric acid to stabilize catecholamines. At the end of thestudy, animals were sacrificed and the brains prepared for histology toverify the site of the microdialysis probe.

Results

Five of the ten animals studied showed the microdialysis probepositioned in the nucleus accumbens (AC). In these five animals (AC-1through AC-5), electrochemical detection revealed a time-dependentchange in molecules associated with enhanced neurotransmission of theserotonin and dopamine systems (FIGS. 4A, B-8A, B). For example, FIG. 4Ais a composite of chromatograms showing changes in monoaminergicmolecules prior to and following CLAV treatment in animals AC-1. Overtime there is an increase in extracellular levels of serotoninconcomitant with a robust increase in the serotonin metabolite5-hydroxyindoleacetic acid (HIAA). While there is a modest decrease indopamine there is a robust increase in its metabolites homovanillic acid(HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC). These increases inmetabolite levels most probably reflect increased neurotransmission ofserotonin and dopamine. However, it is possible that some of themetabolism is not directly coupled to the release of theneurotransmitters (Westernick, 1985).

The percent change from control values for these monoaminergic moleculesfor animals AC-1 through AC-5 are shown in FIGS. 4A, B-8A, B,respectively. The change in serotoninergic molecules was fairlyconsistent. In all cases, animals showed increases in the serotoninmetabolite HIAA of 100%-1000% above baseline (30-50 pm/ml) followingCLAV treatment. The changes in serotonin levels were more variable withthree cases showing an increase of 50%-100% above baseline(undetectable—5 pm/ml), one no change, and the other a modest decrease.The change in dopaminergic molecules was also fairly consistent. In fourof five animals there was an increase in the major dopamine metaboliteHVA of 15%-350% above baseline (50-105 pm/ml) following CLAV treatment.The exception, animal AC-5 (FIG. 8B) showed high baseline levels of HVAthat remained stable over the course of the study; however, levels ofthe other dopamine metabolite DOPAC rose. In three of five animalsdopamine levels increased 50%-450% above baseline (0.5-0.7 pm/ml)following CLAY treatment, while the other two decreased. DOPAC levelsonly rose in two cases AC-3 and AC-5, the same animals that showeddecreases in dopamine.

Animals with the microdialysis probe outside the nucleus accumbensshowed little if any changes in dopamine and serotonin neurotransmissionfollowing CLAY treatment. These sites were the lateral ventricle, bednucleus of stria terminalis, head of the caudate, and ventrolateralthalamus.

Summary

These microdialysis studies indicate CLAY increases serotonin anddopamine neurotransmission in the nucleus accumbens. Recent advances inthe treatment of anxiety disorders have focused on the activation of theserotonin neurotransmission (Feighner 1999). Given the work in this areait is not surprising that CLAV's release of serotonin is accompanied byrobust anti-anxiety behavior in animal models. Interestingly, theenhanced monoaminergic neurotransmission with CLAY treatment mayexplain, in part, the increased sexual arousal in the stress-pronecotton-top tamarin

The activation of serotonin and dopamine neurotransmission in thenucleus accumbens raises the possibility that CLAY may be used to treatdrug addiction, obesity, and schizophrenia. Work by scientists at theNational Institute of Drug Abuse and the National Institute of Diabetesand Digestive and Kidney Diseases have shown that the combinedadministration of amphetamine analogs phentermine and fenfluramine(PHEN/FEN) increases extracellular dopamine and serotonin levels in thenucleus accumbens of rats (Baumann et al., 2000). PHEN/FEN is a aneffective pharmacotherapy for obesity (Weintraub et al., 1984) and inopen clinical trials decreased cocaine craving, alleviated withdrawalsymptoms and prolonged drug abstinence (Rothman et al., 1994).Scientists at the National Institutes of Health concluded drugs with asimilar mechanism to PHEN/FEN causing increased neurotransmission ofserotonin and dopamine in the nucleus accumbens may have utility in thetreatment of substance abuse and obesity.

Work by scientists from Eli Lilly and Company used microdialysis and exvivo tissue analysis of prefrontal cortex and nucleus accumbens toevaluate the mechanism of action of a metabotropic glutamate receptoragonists being developed for the treatment of psychosis (Cartmell etal., 2000a; 2000b). Atypical antipsychotics like risperidone increasedopamine and serotonin neurotransmission in the prefrontal cortex andnucleus accumbens (Cartmell et al., 2000b; Hertel et al. 1997). TheLilly scientists found that activation of metabotropic glutamatereceptors has a similar mechanism elevating DOPAC, HIAA and HVA in thesebrain areas.

How CLAV alters serotonin and dopamine neurotransmission is stillunknown. Neither the monoamine transporters nor the degradative enzymemonoamine oxidase A show binding to CLAV. Antagonism of any of thesereceptor proteins would be expected to alter extracellular levels ofmonoaminergic molecules. It would appear CLAV enhances serotonin anddopamine neurotransmission indirectly by suppressing or activating otherneurotransmitter systems that regulate their activity. CLAV binding toproteolytic enzyme systems that regulate glutamate activity in the brainhave been examined.

Pharmaceutical Formulations and Use

The following formulations are prepared using standard formulationtechniques with a mass ratio of carrier to active compound of about 99:1to about 30:1.

Formulations Dosage β-lactamase inhibitor/dose (mg) Carrier Form I.Clavulanic acid, potassium/30 starch/maltose capsule II. Clavulanicacid, sodium/50 microcrystalline tablet cellulose/trehalose III.Tazobactam/75 saline injectable IV. Tazobactam/125 starch microspheresinjectable V. Clavulanic acid, potassium/150 saline injectable VI.Sulbactam/200 saline injectable VII. Sulbactam/250 polylactideinjectable microspheres VIII. Cefsulodin/50 starch microspheresinjectable IX. Moxalactam bis-indanyl ester/20 microcrystalline capsulecellulose X. Cefazolin/120 saline injectable XI. Cefazolin sulfoxide/10saline injectable XII. Cephalexin sulfone/35 polylactic acid injectablemicrospheres XIII Cefaclor sulfoxide/75 saline injectable

Formulation Use

A). A male patient suffering from erectile dysfunction is administered adose of Formulation I about 1 hour before conjugal activity to improvesexual performance.

B). A female patient suffering from mild depression self-administersFormulation IX t.i.d. over a 2-week period to improve her libido.

C). A dog breeder administers Formulation V to his female and/or malecanines to promote breeding activities.

D). A zoo keeper administers Formulation XI to male and female partnersof a rare simian species to promote reproductive breeding of theanimals.

1. A method for improving sexual function in a mammal suffering from ordisposed to develop sexual dysfunction or otherwise in need of enhancedsexual function, said method comprising the step of administering tosaid mammal a β-lactam compound capable of inhibiting the activity of abacterial enzyme selected from β-lactamase or penicillin binding proteinor a carboxypeptidase, where the β-lactam compound is administered in anamount therapeutically effective to treat the sexual dysfunction orenhance the sexual function of said mammal. 2.-31. (canceled)